Chemiluminescent acridinium compounds are extremely useful labels that have been used extensively in immunoassays and nucleic acid assays. A recent review, Pringle, M. J., Journal of Clinical Ligand Assay vol. 22, pp. 105-122 (1999) summarizes past and current developments in this class of chemiluminescent compounds.
Seminal work by McCapra, F. et al., Tetrahedron Lett. vol. 43, pp. 3167-3172 (1964) and Rahut et al. J. Org. Chem vol. 301, pp. 3587-3592. (1965) disclosed that chemiluminescence from phenyl esters of acridinium salts could be triggered by alkaline peroxide. Since these early studies, interest in acridinium compounds has increased because of their utility as chemiluminescent labels. The application of the acridinium ester, 9-carboxyphenyl-N-methylacridinium bromide in an immunoassay was disclosed by Simpson, J. S. A. et al., Nature vol. 279, pp. 646-647 (1979). This acridinium ester is quite unstable owing to hydrolysis of the ester linkage between the acridinium ring and the phenol thereby limiting its commercial utility unless special precautions are taken to protect the ester linkage from hydrolysis. For example Arnold et al. in U.S. Pat. No. 4,950,613 have shown that the hydrolytic stability of unstable acridinium esters can be alleviated somewhat with additives.
Different strategies for increasing the hydrolytic stability of acridinium compounds by altering their structures have been described. Law et al., Journal of Bioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989) reported that phenols containing two methyl groups flanking the phenolic group afforded acridinium esters that are more resistant to hydrolysis. The acridinium ester DMAE-NHS [2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl 10-methylacridinium-9-carboxylate] was found to have the same light output as an acridinium ester lacking the two methyl groups but was significantly more resistant to hydrolysis. U.S. Pat. Nos. 4,918,192 and 5,110,932 describe DMAE and its applications. U.S. Pat. No. 5,656,426 to Law et al. discloses a hydrophilic version of DMAE termed NSP-DMAE-NHS ester where the methyl group on the acridinium ring nitrogen is replaced with a sulfopropyl group. Natrajan et al. in U.S. Pat. No. 6,664,043 B2 disclosed NSP-DMAE derivatives with hydrophilic modifiers attached to the phenol.
A different class of stable chemiluminescent acridinium compounds has been described by Kinkel et al., Journal of Bioluminescence and Chemiluminescence vol. 4, pp. 136-139 (1989) and Mattingly, Journal of Bioluminescence and Chemiluminescence vol. 6, pp. 107-114 (1991) and U.S. Pat. No. 5,468,646. In this class of compounds, the phenolic ester linkage is replaced by a sulfonamide moiety, which is reported to impart hydrolytic stability without compromising the light output. The structure of DMAE-NHS and the generalized structure of an acridinium sulfonamide are illustrated in FIG. 1 along with the numbering system commonly used for acridinium ester. The phenol and the sulfonamide moieties are also commonly referred to as leaving groups. Chemiluminescent acridinium compounds containing other leaving groups such as oximes have also been disclosed. See Renotte et al. Luminescence 2000, 15, 311-320.
Acridinium compounds, in aqueous solution, exist in equilibrium with adducts formed by the addition of water to C-9 of the acridinium ring. This adduct is commonly referred to as the pseudobase. The acridinium-pseudobase equilibrium, which is illustrated in FIG. 2, is strongly influenced by the pH of the aqueous medium. In acidic solutions the acridinium form is favored whereas in basic solution, the predominant form is the pseudobase. The acridinium-pseudobase equilibrium is also affected by the structure of the acridinium compound. Acridinium esters containing electron-donating groups at C-2 and/or C-7, reduce the electrophilicity of C-9 and raise the pH at which the transition from the acridinium form to the pseudobase takes place. Acridinium sulfonamides also are less prone to pseudobase formation than acridinium esters.
Chemiluminescence from acridinium compounds is normally triggered with hydrogen peroxide. The mechanism of light emission is believed to involve addition of hydrogen peroxide to C-9 of the acridinium ring followed by cleavage of the leaving group and concomitant formation of a high energy, dioxetanone intermediate. Rapid decomposition of the dioxetanone intermediate is presumed to lead to formation of the acridone in an electronically excited state. Light emission occurs when the acridone in the excited state reverts to the ground state. The formation of the dioxetanone intermediate has not been demonstrated conclusively and, a recent theoretical study postulates that cleavage of the leaving group and formation of the excited state acridone may occur simultaneously. (Rak et al. J. Org. Chem. 1999, 64, 3002-3008).
In practice, light emission from acridinium compounds and their conjugates using hydrogen peroxide is normally accomplished by an initial treatment with aqueous acid to effect complete conversion of the pseudobase to the acridinium form followed by the addition of aqueous base. Acid treatment is necessary because the pseudobase cannot react with hydrogen peroxide. The length of acid treatment and the strength of the acid that must be used depend upon the structure of the acridinium compound. The addition of base ionizes the hydrogen peroxide molecule to form the hydroperoxide ion, which then adds to C-9 of the acridinium ring and initiates light emission. For convenience, hydrogen peroxide is often added along with the aqueous acid as a single reagent. Typically, light emission from the acridinium compound or its conjugate occurs over a time period of a few seconds.
The kinetics or the rate of light emission from the acridinium compound or its conjugate depends on a number of factors. Both the concentrations of hydrogen peroxide as well as that of the base can affect the duration of light emission. The presence of surfactants can also affect the rate of light emission as well as the quantum yield. In addition, the structure of the acridinium compound also has a profound effect on the kinetics of light emission. Although substituents on the acridinium ring, at the acridinium nitrogen and on the leaving group can all affect the kinetics of light emission, the impact of various structural features of the leaving group on light emission has been most widely reported. For example, Adamczyk et al. (Tetrahedron 1999, 55, 10899-10914) from a study of various acridinium sulfonamides have shown that the kinetics of light emission can be varied by structural variation of the sulfonamide leaving group without affecting the total amount of light emitted by these compounds. These investigators concluded from their study that steric factors were more influential in varying the rate of light emission than the pKa of the sulfonamide leaving group. Increasing steric congestion at the acridinium sulfonamide nitrogen led to slow light emission while relieving such steric hindrance speeded up light emission.
A similar study on acridinium phenyl esters was reported by Nelson et al. (Biochemistry 1996, 35, 8429-8438). From their study, the authors concluded that the pKa of the phenol leaving group had a more significant impact on the kinetics of light emission than steric effects. Electron withdrawing groups on the phenyl ring led to an acceleration in the rate of light emission while electron-donating groups led to a suppression of the rate. The acridinium esters described by Nelson are quite susceptible to hydrolysis even though they show fast light emission. The ‘hybridization protection assays’ described by Nelson, in fact, take advantage of the fact that acridinium esters conjugated to nucleic acid probes and, not hybridized to their targets, can be hydrolyzed at much faster rates that the labeled hybridized probes.
Steric effects also play an important role in the kinetics of light emission of acridinium phenyl esters. For example Woodhead et al. in U.S. Pat. No. 5,656,207 have reported that the kinetics of light emission of an acridinium ester containing methyl groups at the 2′ and 6′ carbon atoms on the phenol can be differentiated from an analogous acridinium ester lacking these two substituents by a careful selection of the light triggering reagents.
Acridinium esters containing methyl groups at the 2′ and 6′ carbons emit light more slowly than acridinium esters lacking these substituents. On the other hand, the presence of the methyl groups imparts much greater hydrolytic stability on the acridinium ester as noted by Law et al., (Journal of Bioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989) who compared the hydrolytic stability of DMAE-NHS with an analogous acridinium ester lacking the 2′ and 6′ methyl groups. Hydrolytic stability is important especially for commercial applications of acridinium esters in automated immunochemistry instruments because it is tied to reagent stability. Reagents with a long shelf are often preferred because they cause less day-to-day variations in assay performance and do not create as much waste.
Hydrolytically stable acridinium esters such as NSP-DMAE described in U.S. Pat. No. 5,656,426 when conjugated to proteins or small molecules, typically emit light over a period of five seconds when their chemiluminescence is triggered with alkaline peroxide.
In view of the foregoing, there is a need in the art for acridinium esters which exhibit both hydrolytic stability and fast light emission. It is therefore and object of the invention to provide acridinium esters which are hydrolytically stable, e.g., comparable to NSP-DMAE, but which also exhibit much faster light emission, that is, on the order of one to two seconds.